The studies in this application seek to elucidate the control of neuroendocrine differentiation in normal calcitonin producing thyroid C- cells and their neoplastic counterpart, medullary thyroid carcinoma (MTC). Understanding this control could provide important insight for events in tumor development and progression. Medullary thyroid carcinoma is a good model for this study, since defined stages of progression which involve loss of neuroendocrine differentiation features including capacity for calcitonin production, have been identified. We have developed an in vitro system to study steps of MTC differentiation and progression. MTC cells in culture can be differentiated with respect to many characteristics of normal C-cells including increased calcitonin gene expression, either chemically or by ras oncogene introduction. The current proposal will further characterize these findings, by cell biology, biochemistry, and molecular biology, concentrating on the molecular mechanisms regulating the induced differentiation. The studies proposed will be important not only for identifying the processes which may be involved in neuroendocrine differentiation, and for further elucidating the mechanisms of ras oncogene function in cells. 1. The role of endogenous ras oncogenes in MTC cell differentiation will be investigated by performing several maneuvers to block ras gene function. In addition, the levels of endogenous ras protein expression in normal and hyperplastic C cells, as well as well-differentiated and poorly differentiated MTC, will be examined. Second, it will be determined whether the signal transduction alterations detected following ras gene insertion in MTC cells, are sufficient to induce all or part of the differentiated phenotype mediated by ras. The generality of ras-mediated differentiation in MTC cells will be examined, using two new human MTC cell lines, to determine whether subtypes of MTC exist, differing in their response to ras. Such subtypes may identify other genetic events involved in C-cell differentiation or MTC tumor progression. 2. A ras-responsive transcriptional element in the calcitonin gene will be further characterized, by continuing our isolation of the DNA binding proteins and their genes. Regulation of expression of these proteins, as well as their function in differentiation of MTC and other cells in vitro, and in vivo, will be examined.